Biosensor having decoupled capture chamber and detection chamber, using particle aggregation and size-separation

ABSTRACT

A biosensor using a decoupled microfluidic device, which has a capture chamber and a detection chamber separate from and in fluid communication with each other. The sensing method is based on particle aggregation via homogeneous reactions, by employing microparticles having antibodies on their surfaces which can form aggregates through antigen mediation. Either size-separation or magnetic based separation is used to separate aggregates from single microparticles; the aggregates are later dissociated and the resulting single microparticles are counted to measure the amount of the antigen. Another biosensor uses a decoupled microfluidic device with a capture chamber and a detection chamber, and a 3-D structure in the capture camber to increase immobilized antibody concentration. Immunoreaction efficiency is improved by increasing the number of antibody per reaction volume in the capture chamber.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to biosensor for point-of-care (POC) diagnostics,and in particular, it relates to a biosensor using a decoupledmicrofluidic device that employs particle aggregation andsize-separation.

Description of Related Art

High-sensitive, rapid and inexpensive biosensors, i.e. sensors fordetecting biomolecules, such as DNA or protein sensors, are desired forpoint-of-care (POC) diagnostics. In conventional biosensors, the capturestep and the detection step are coupled to each other. In the capturestep, the biomolecules to be detected are captured by capture agents ina chamber of the biosensor; in the detection step, the capturedbiomolecules are detected. In the case of such capture and detectioncoupled biosensor, there is often a tradeoff between one (e.g. capture)and the other (e.g. detection) and both cannot be optimizedsimultaneously.

A team at Stanford Genome Technology Center developed an ultra-sensitiveelectronic microfluidic technique, referred to as “decoupled” digitaldetection. Using this technique, the team has demonstrated an attomolarlevel limit of detection (LOD) with palm-top size devices. See Mok etal., Digital microfluidic assay for protein detection, Proc Natl AcadSci USA 2014 Feb. 11; 111(6):2110-5 (“Mok et al., PNAS 2014”); see alsoUS 2011/0312518 A1. In this decoupled system, the capture chamber andthe detection chamber are completely separated physically, which offersa large degree of flexibility in tailoring each chamber. Noises due tonon-specific binding can be reduced, resulting in increased sensitivity.However, this technology is slow, requiring a relatively long time forthe capture reaction (on the order of one hour) due to the lowconcentration of capture antibodies and the small volume of the capturechamber.

SUMMARY

The present invention is directed to a biosensor and related method thatsubstantially obviates one or more of the problems due to limitationsand disadvantages of the related art.

An object of the present invention is to improve reaction speed of thedecoupled biosensor.

Additional features and advantages of the invention will be set forth inthe descriptions that follow and in part will be apparent from thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the structure particularly pointed out in the writtendescription and claims thereof as well as the appended drawings.

To achieve these and/or other objects, as embodied and broadlydescribed, the present invention provides a microfluidic device forminga biosensor, which includes: a capture chamber; a detection chamberseparate from and in fluid communication with the capture chamber, thedetection chamber having a detection device for detecting singlemicroparticles passing through the detection chamber; and a separationmechanism located in the capture chamber or between the capture chamberand the detection chamber, for separating aggregations of themicroparticles and single microparticles.

In another aspect, the present invention provides a microfluidic deviceforming a biosensor, which includes: a capture chamber; a detectionchamber in fluid communication with the capture chamber, the detectionchamber having a detection device for detecting single microparticlespassing through the detection chamber; and a magnetic based separationmechanism located in the capture chamber, including a magnet forgenerating a magnetic field in the capture chamber.

In yet another aspect, the present invention provides a method fordetecting an antigen using a biosensor, the biosensor having a capturechamber and detection chamber which are separate from and in fluidcommunication with each other, the method including: applying a samplein the capture chamber, the sample containing the antigen andmicroparticles having antibodies immobilized on their surfaces; allowingthe microparticles to form aggregates by antigen mediation in thecapture chamber; separating single microparticles and the aggregates byretaining only the aggregates in the capture chamber; dissociating theaggregates into single microparticles; and detecting, in the detectionchamber, an amount of single microparticles obtained from dissociatedaggregates.

In yet another aspect, the present invention provides a method forsimultaneously detecting first and second antigens using a biosensor,the biosensor having a capture chamber and detection chamber which areseparate from and in fluid communication with each other, the methodincluding: applying a sample in the capture chamber, the samplecontaining the first and second antigens and first and secondmicroparticles having antibodies immobilized on their surfaces; allowingthe first and second microparticles to form respective first and secondaggregates mediated by the first and second antigens, respectively, inthe capture chamber; separating single first and second microparticlesand the first and second aggregates by retaining only the first andsecond aggregates in the capture chamber; dissociating the first andsecond aggregates into single first and second microparticles by usingan elution buffer; and detecting, in the detection chamber, an amount ofsingle first and second microparticles obtained from dissociated firstand second aggregates.

In yet another aspect, the present invention provides a microfluidicdevice forming a biosensor, which includes: a capture chamber, having athree-dimensional structure and antibodies immobilized on thethree-dimensional structure; and a detection chamber separate from andin fluid communication with the capture chamber, the detection chamberhaving a detection device for detecting single microparticles passingthrough the detection chamber.

In yet another aspect, the present invention provides a microfluidicdevice forming a biosensor, which includes: a capture chamber; and adetection chamber separate from and in fluid communication with thecapture chamber, the detection chamber having a detection device fordetecting single microparticles and dimerized microparticles passingthrough the detection chamber, the detection device generating differentsignals upon detecting the single microparticles and the dimerizedmicroparticles.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A) and 1(B) schematically illustrate a biosensor device andmethod using homogeneous capture (aggregation) and size separationaccording to an embodiment of the present invention.

FIGS. 2(A)-(D) schematically illustrate variations of the embodiment ofFIG. 1.

FIG. 3 schematically illustrates a biosensor device and method usingcross flow filtration according to another embodiment of the presentinvention.

FIG. 4 schematically illustrates a biosensor device and method usingroundtrip flow filtration according to another embodiment of the presentinvention.

FIGS. 5(A) and 5(B) schematically illustrate a biosensor device andmethod using homogeneous capture and magnetic separation according toanother embodiment of the present invention.

FIGS. 6(A)-6(B) schematically illustrate biosensor devices and methodsaccording to other embodiments of the present invention, which implement3-dimensional structures for antibody immobilization.

FIGS. 7(A)-(C) schematically illustrate a biosensor device and methodaccording to another embodiment of the present invention.

FIGS. 8(A) and 8(B) schematically illustrate a biosensor device andmethod according to yet another embodiment of the present invention,which is capable of detecting multiple types of antigens simultaneouslypresent in a sample.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the present invention provide methods to improveimmunoreaction efficiency by increasing the number of antibody perreaction volume in decoupled biosensors, which have a capture chamberand a separate detection chamber. In some embodiments of the presentinvention, in the capture chamber immunoreaction can occur under acondition of more than 5×10{circumflex over ( )}−7 mol/L captureantibody concentration.

Two types of reactions may be used in the capture step, namelyhomogeneous reactions and heterogeneous reactions, described in moredetail later. The embodiments shown in FIGS. 1(a)-5(B) use homogeneousreactions, and the embodiment shown in FIGS. 6(A)-6(B) use heterogeneousreactions.

The embodiments shown in FIGS. 1(A)-4 implement particle aggregation andsize-separation mechanism in the capture chamber of the decoupledbiosensor. The decoupled biosensor in this invention has asize-separation moiety between the capture chamber and the detectionchamber.

To achieve aggregation, two or more types of antibodies can be used; orin an alternative embodiment, one type of antibody can be used. In thelatter case (one type of antibody), the antigen should have more thantwo epitopes or should form multi-mers. The antibodies are immobilizedonto microparticles, typically with more than two antibodies permicroparticle. In the presence of the antigen, the microparticles formaggregations through antigen mediation. This aggregation reaction isconducted in the capture chamber of the decoupled biosensor. Afteraggregation reaction in the capture chamber, reaction samples aretransferred to the detection chamber. If the aggregation happens and theaggregation size is larger than the threshold which can pass through thesize-separation mechanism, the aggregation is trapped and cannot passthrough to the detection chamber. Then by using an elution buffer whichcan dissociate aggregated microparticles, aggregations are dissociatedinto single microparticles which can pass through the size-separationmechanism and go to the detection chamber. By detecting the dissociatedsingle microparticles one-by-one using a device to measure a change inelectrical impedance, the amount of antigen can be quantified orestimated.

In the embodiments shown in FIG. 5, instead of the size-separationmechanism, magnetic force is used to separate bound (aggregated) andunbound (single) microparticles. In this case, two kinds ofmicroparticles are used, including non-magnetic microparticles(microparticles that are not attracted by magnetic field) and magneticmicroparticles which are attracted by magnetic field. A magnetic fieldis established in an area between the capture chamber and detectionchamber. Different antibodies can be used for the non-magneticmicroparticles and the magnetic microparticles respectively, or the sameantibody can be used if the antigen has more than two epitopes or formsmulti-mer. In the presence of the antigen, non-magnetic microparticlesand magnetic microparticles form aggregations through antigen mediation.This aggregation reaction is conducted in the capture chamber of thedecoupled biosensor. After aggregation reaction in the capture chamber,aggregated microparticles and unbound magnetic microparticles aretrapped by the magnetic field, although unbound non-magneticmicroparticles can continue downstream. Then by using an elution bufferwhich can dissociate aggregated microparticles, aggregations aredissociated into single microparticles, and dissociated non-magneticmicroparticles go to the detection chamber. Magnetic microparticlesremain in the magnetic field.

The microparticles may be made of a suitable material that can causechange in the electrical properties of the fluid so that they can bedetected by the detection device in the detection chamber. Examplesinclude polymer particle, such as polystyrene particles or beads, metalcolloids, etc. Methods for making such microparticles are known in thefield. See, for example, US 2011/0312518, para. [0140].

The embodiments are described in more detail below with reference to thedrawings.

FIGS. 1(A) and 1(B) schematically illustrate a biosensor device andmethod based on homogeneous capture (aggregation) according to a firstembodiment of the present invention. This embodiment uses sizefiltration to separate bound and unbound microparticles. As shown inFIG. 1(A), the biosensor is a microfluidic device which has a capturechamber 11 with an inlet 13, a detection chamber 12, and a waste channel15 with an outlet (not shown). The detection chamber 12 is a microchannel where only single microparticle can pass through, and has avoltage applied to its two ends to measure a change in electricalimpedance, forming a single particle counter 16, similar to thatdescribed in Mok et al. PNAS 2014, and US 2011/0312518 (see [0146],[0184], [0298]-[0303], FIGS. 4, 26, 27). A filter 14 having apredetermined pore size is provided between the capture chamber 11 andthe detection chamber 12; the filter can pass single microparticles butstops aggregated microparticles.

FIGS. 1(A) and 1(B) illustrate five steps of the detection method. InStep 1, a sample solution containing an antigen 1 (biomolecules to bedetected, also referred to as analyte) and two types of microparticles2A and 2B is injected via the inlet 13 into the capture chamber 11 ofthe biosensor. The two types of microparticles 2A and 2B have tworespective types of antibodies attached on their surfaces; both types ofantibodies bind to the antigen 1. The two types of microparticlesthemselves may be the same except for the different antibodies. Theconcentration of microparticles in the sample is controlled so as togive a satisfactory reaction rate. As mentioned earlier, an antibodyconcentration of 5×10{circumflex over ( )}−7 mol/L is desired so thatthe reaction in the capture chamber can be substantially completedwithin 10 min.

Note that in this embodiment, the microparticles with antibodies aremixed in the sample solution injected into the biosensor at the time ofuse. Alternatively, microparticles with antibodies can be provided inthe capture chamber before sample injection; i.e., the biosensors thatare provided to the users already contain microparticles with antibodiesin the capture chamber. The alternative approach is more convenient forthe users, but the antibodies' stability might be a concern.

In Step 2, microparticles (both types 2A and 2B) bind to antigens 1 andform aggregated complexes 3. This type of reaction, where theantibodies, being attached to the microparticles, are free in the samplesolution (as opposed to being immobilized in the capture chamber), isreferred to as a homogenous reaction. The reaction is allowed to proceedfor a period of time. In Step 3, a wash solution 4 is applied to thecapture chamber 11 to wash unbound microparticles through the filter 14.The pore size of the filter 14 is designed so that unbound singlemicroparticles 2A, 2B pass through the filter while aggregated complexes3 formed by multiple microparticles do not and are captured by thefilter.

Then, in Step 4, an elution buffer 5 is injected into the capturechamber 11 via the inlet 13. The elution buffer 5 contains substancesthat dissociate the aggregated microparticles 3, and the resultingsingle microparticles 2A, 2B pass through the filter 14. In Step 5, thedissociated microparticles 2A and 2B, having passed through the filter14, are counted in the detection chamber 12 by the single particlecounter 16.

The technique employed in this embodiment solves the challenge of how toseparate bound and unbound microparticles.

The antibodies on the microparticles are typically an order of 100 timessmaller than the microparticles. Multiple antibodies are typicallypresent on the surface of each microparticle. Because each of the twotypes of microparticles can bind to the antigen (at two respective sitessimultaneously), the aggregate of microparticles tend to form longchains, which can have branches. In the detection step (Step 5), thereis no need to discriminate between the two types of microparticles 2Aand 2B. Although there is not an exact relationship between the totalnumber of single microparticles counted in Step 5 and the number ofantigens, the amount of antigens can be estimated from the number ofmicroparticles counted.

FIGS. 2(A)-2(D) schematically illustrate variations of the firstembodiment, using size filtration for separation. FIG. 2(A) shows adevise using a filter 14 to separate aggregated and un-aggregatedmicroparticles, as in the embodiment of FIG. 1. FIG. 2(B) shows a deviceusing a continuously tapered channel 17 between the capture chamber 11and the detection chamber 12 for separating the aggregated andun-aggregated microparticles. FIG. 2(C) shows a device using a step-wisetapered channel 18 between the capture chamber 11 and the detectionchamber 12 for separating the aggregated and un-aggregatedmicroparticles.

FIG. 2(D) shows a device using a cascade of filters 14A-C graduallyreducing in pore size for separating the aggregated and un-aggregatedmicroparticles. In the structure of FIG. 2(D), the first filter 14A(largest pore size) allows some smaller aggregated microparticles topass through, while the last filter 14C (smallest pore size) only allowssingle microparticles to pass through. This design reduces clogging ofthe filters.

The operations of the variations shown in FIGS. 2(A)-2(D) are similar tothe embodiment shown in FIG. 1(A), where the tapered shapes 17, 18 andthe cascading filters perform similar functions as the filter in FIG.1(A).

FIG. 3 schematically illustrates a biosensor device and method accordingto another embodiment of the present invention, based on homogeneousreaction and size filtration, using cross flow filtration. Themicrofluidic device has a capture chamber 31 which has a looped shape. Avalve A is located along the loop path of the capture chamber 31, nearthe inlet 33. A filter 34 is provided between the capture chamber 31 anda transfer channel 36; unbound (singe) microparticles 2A, 2B can passthrough the filter 34 while aggregated complexes 3 cannot. The filter 34may be provided at a wider interface area between the capture chamber 31and the transfer channel 36 to increase the filter area. The transferchannel 36 has two branches; one branch is controlled by a valve B andleads to a waste reservoir 35, and the other is controlled by a valve Cand leads to the detection chamber 32.

In operation, after a sample solution containing the antigen and the twotypes of microparticles are introduced to the capture chamber 31 via theinlet 33, with valve A open and valves B and C closed, theimmunoreaction is allowed to proceed for a period of time. Then, withvalve A open, valve B open, and valve C closed, a wash solution isapplied to the capture chamber 31 to wash unbound microparticles throughthe filter 34, so as to separate aggregated microparticles and singlemicroparticles. The fluid flows in the looped part of the capturechamber 31, in a counter-clockwise direction in this example, and theunbound microparticles flow through the filter 34 and is discharged viavalve B into the waste reservoir 35 (as schematically indicated by thedashed-line arrows in FIG. 3).

Thereafter, valve A is closed, valve B is closed, and valve C is open;an elution buffer is injected into the capture chamber 31 via the inlet33 and flows through it in the looped part of the capture chamber, inthe clockwise direction in this example. The aggregated microparticlesare dissociated, and the dissociated microparticles pass though valve Cto the detection chamber 32 to be detected (as schematically indicatedby the solid-line arrows in FIG. 3).

In this embodiment, the filter 34 is oriented along a side plane of thelooped part of the capture chamber so that when the fluid circulates inthe looped part of the capture chamber 31, it flows in a substantiallyparallel direction over the surface of the filter, which helps to flushthe filter 34 to prevent clogging, and promotes the capture reaction.Cross flow filtration methods per se are generally known.

FIG. 4 schematically illustrates a biosensor device and method accordingto another embodiment of the present invention, based on homogeneousreaction and size filtration, using a roundtrip flow. The microfluidicdevice has a capture chamber 41 with two filters 44A and 44B at its twoends. At each end of the capture chamber 41, beyond the respectivefilter, an inlet 43A/43B and an outlet branch 45A/45B are provided, witha valve (A and B, respectively) that opens/closes the respective inletand outlet. The inlet at each end is for supplying a flow solution andthe outlet at each end is for waste discharge. A transfer channel 46 isjoined to the capture chamber 41 between the two filters 44A/44B,controlled by a valve C, and leads to the detection chamber 42.

In operation, after a sample solution containing the antigen and the twotypes of microparticles are introduced to the capture chamber 41 via oneof the inlets, with valve C closed, the immunoreaction is allowed toproceed for a period of time. Then, valve C remains closed, and valves Aand B are operated to alternately open and close the inlet 43A/43B andoutlet 45A/45B at the two ends, and a wash solution is introducedthrough the two inlets. More specifically, during first time periods,valve A opens its associated inlet 43A and close its associated outlet45A while valve B closes its associated inlet 43B and opens itsassociated outlet 45B, and the wash solution flows from left to right inthe illustrated example; and during second time periods which alternatewith the first time periods, valve A closes its associated inlet 43A andopens its associated outlet 45A while valve B opens its associated inlet43B and closes its associated outlet 45B, and the wash solution flowsfrom right to left. This creates a roundtrip flow pattern, and unboundmicroparticles are discharged with the wash solution via the outlets 45Aand 45B. As a result, the aggregated microparticles and singlemicroparticles are separated by the filters 44A and 44B. The roundtripflow of the fluid in the capture chamber 41 helps to flush the filters44A and 44B to prevent clogging.

Thereafter, valves A and B are controlled to close both outlets 45A and45B, and to open one or both of the two inlets 43A and 43B, and valve Cis open; an elution buffer is injected into the capture chamber via theopen inlet(s). The aggregated microparticles are dissociated, and thedissociated microparticles pass though valve C to the detection chamber42 to be detected.

FIGS. 5(A) and 5(B) schematically illustrate a biosensor device andmethod based on homogeneous capture and magnetic separation according toanother embodiment of the present invention. As shown in FIG. 5(A), themicrofluidic device is similar to that shown in FIG. 1(A), except thatno filter is provided, but a magnetic field is applied to an area of thecapture chamber 11 near the detection chamber 12, for example by using amagnet 54.

FIGS. 5(A) and 5(B) illustrate five steps of the detection method ofthis embodiment. In Step 1, a sample solution containing an antigen 1and two types of microparticles 2C and 2D is injected via the inlet 13into the capture chamber 11. The two types of microparticles have tworespective types of antibodies attached on their surfaces; both types ofantibodies bind to the antigen. One type of microparticles 2D ismagnetic, the other type 2C is non-magnetic.

In Step 2, microparticles (both types, 2C and 2D) bind to antigens 1 andform aggregated complexes 3. The reaction is allowed to proceed for aperiod of time. In Step 3, a wash solution 4 is applied to the capturechamber 11 to wash unbound non-magnetic microparticles 2C out of thecapture chamber. Both the aggregate microparticle complexes 3, whichcontain the magnetic type of microparticles 2D, and the unbound magneticmicroparticles 2D are retained in the area of the capture chamber 11where the magnetic field is present.

Then, in Step 4, an elution buffer 5 is injected into the capturechamber 11 via the inlet 13. The elution buffer 5 dissociates theaggregated microparticles 3. The dissociated non-magnetic microparticles2C are no longer retained by the magnetic field. In Step 5, thenon-magnetic microparticles 2C flow through the detection chamber 12 andare counted by the single particle counter 16. The amount of antigens 1can be estimated from the number of microparticles counted.

The magnetic microparticles 2D remain in the capture chamber 11 in Step5; they can be released later by removing the magnetic field.

FIGS. 6(A)-6(B) schematically illustrate a biosensor device and methodaccording to other embodiments of the present invention, which implement3-dimensional structures for antibody immobilization within thedetection chamber. The 3-d structure can be a polymer material (FIG.6(A)) or plurality of micro size tubes (FIG. 6(B)). By using 3-dstructure for antibody immobilization, the number of antibodies forimmunoreaction per volume within the capture chamber can be increased.

FIGS. 6(A)-6(B) show a part of the capture chamber of a decoupledbiosensor of these embodiments. The other parts of the biosensor,including the detection chamber, are similar to those shown in theembodiment of FIG. 1. The capture chamber 61 has antibodies 63immobilized in a volume of the capture chamber, forming a 3-d structure.

In FIG. 6(A), the 3-d structure is formed by a polymer material 62immobilized in the capture chamber 61, and attached along the polymerchains are antibodies 63 which bind to the antigen 1. The polymermaterial 62 may be any suitable solid polymers, such as hydrophilicpolymer, carboxyl methyl dextran (CMD), polyethylene glycol (PEG),poly(acrylic acid), poly(methacrylic acid), etc. Using the polymers 62in the capture chamber 61 increases the antibody binding sites pervolume. No filter is required between the capture chamber and thedetection chamber. In operation, a sample containing the antigen 1 andmicroparticles 2 (only one type is needed) with antibodies is introducedto the capture chamber 61, as shown in FIG. 6(A). The antibodies on themicroparticles 2 and the antibodies on the polymer material 62 bind totwo different sites of the antigen 1. Through antigen mediation, themicroparticles are captured by the antibodies 63 immobilized onto the3-d structure 62 in the capture chamber 61. This type of reactions,where antibodies 63 are immobilized in the capture chamber 61, isreferred to as heterogeneous reactions. After a period of time, a washsolution is applied to the capture chamber 61 to wash away the unboundmicroparticles 2, and then an elution buffer is added to the capturechamber 61 to dissociate the microparticles 2 from the polymer 62. Thedissociated single microparticles 2 move to the detection chamber to becounted, so that the amount of antigen 1 can be quantified or estimated.

In FIG. 6(B), the 3-D structure of the capture chamber 61 is formed byforming a plurality of micro tubes 64, with antibodies 63 immobilized onthe surface of the micro tubes. The diameter of the micro tubes 64should be larger than the microparticles 2. Preferably, the total volumeof the micro tubes 64 depends on the sample volume. This structureincreases the antibody binding sites per volume of the capture chamber61. No filter is required between the capture chamber 61 and thedetection chamber. The operation of this biosensor is otherwise similarto the one in FIG. 6(A).

FIGS. 7(A)-(C) schematically illustrate a biosensor device and methodaccording to another embodiment of the present invention, which usesmicroparticles to form dimers. Two types of antibodies are used; eachmicroparticle has only one antibody on it, either the first type or thesecond type. I.e., there are two types of microparticles, and they formdimmers (one of each type of microparticle) in the presence of theantigen. FIG. 7(A) schematically shows two microparticles of two types,2E and 2F, and the antigen 1. FIG. 7(B) schematically shows the dimerthey form. Alternatively (not shown), the antigen 1 is one that has twoepitopes or form dimers, and only one type of microparticle with onetype of antibody is used; two microparticles each having one antibodyform a dimer in the presence of the antigen. This is a type ofhomogeneous reaction where the antibodies are free in the samplesolution.

The biosensor of this embodiment has a structure similar to that shownin FIG. 1(A), having a capture chamber and a detection chamber, butwithout any filter between the two chambers. As shown in FIG. 7(C), thedetection chamber 72 is sized so that the dimers can pass through it,and the single particle counter is designed so it can discriminatebetween single microparticles and dimerized microparticles based on thepulse width of the electrical signal. The number of antigens 1 can bequantified based on the number of dimers detected. This embodiment doesnot require size separation.

FIGS. 8(A) and 8(B) schematically illustrates another embodiment of thepresent invention, which is capable of detecting multiple types ofantigens simultaneously present in a sample. Multiple types ofmicroparticles are provided, where the different types of microparticleshave different antibodies on them that bind to different types ofantigens. The different types of microparticles also affect electricalimpedance differently in the single particle counter of the detectionchamber. For example, they may generate pulses of different magnitudesin the detected electrical current.

The working principle with respect to each antigen is similar to that ofthe embodiment of FIG. 1(A). In other words, for each antigen, two typesof microparticles are provided in the sample, and they aggregate in thepresence of that antigen. FIG. 8(A) schematically illustrates threedifferent antigens and respective different types of microparticles.Note that for simplicity, the two types of microparticles for eachantigen are not differentiated in this drawing. The elution buffer usedcontains substances that can dissociate all microparticles from theirrespective antigens.

The two types of microparticles that correspond to the same antigen giverise to the same electrical signal. Microparticles that correspond todifferent antigens give rise to different electrical signals, such aspulses of different magnitude (see FIG. 8(B)). Thus, the number ofsingle microparticles corresponding to each antigen can be separatelycounted. Using this technique, multiplexing biosensing (detection ofmultiple antigens in the same sample) can be realized.

The structure of the biosensor in the embodiment of FIG. 8(A) is similarto that of the embodiment of FIG. 1(A), with a capture chamber 11, adetection chamber 12, and a filter separating 14 the two. Themultiplexing technology can also be applied to the embodiments shown inFIGS. 2(A)-7; in fact, it can be applied to any microfluidic biosensorswith decoupled capture chamber and detection chamber, including thatdescribed in the Mok et al., PNAS 2014 article, which uses the type ofsingle particle counter that can differentiate the different types ofmicroparticles.

In practice, the reaction rate of an immunoreaction can be calculated asfollows:

Reaction  rate  (%):  X/[Ag₀] * 100$X = \frac{\alpha*{\beta\left( {1 - {\exp\left( {a*k_{on}*t} \right)}} \right)}}{\left. {\beta - {\alpha*{\exp\left( {a*k_{on}*t} \right)}}} \right)}$p = [Ab] + [Ag] + k_(off)/k_(on) q = [Ab] * [Ag]$a = \sqrt{p^{2} - {4q}}$ α = (p + a)/2 β = (p − a)/2In one example, the following parameters are used in the calculation:antigen (Ag) concentration=1 ng/mL; Ag molecular weight=50 kDa;k_(on)=10{circumflex over ( )}6; k_(off)=10{circumflex over ( )}−4;antibody [Ab] concentration>5×10{circumflex over ( )}−7 mol/L. Thisantibody concentration is the concentration above which almost 100% ofthe antigens are captured within 10 min (using ka=10{circumflex over( )}6, kd=10{circumflex over ( )}−4). It can be seen that antibodyconcentration plays an important role in increasing reaction speed inthe capture chamber of a biosensor.

It will be apparent to those skilled in the art that variousmodification and variations can be made in the biosensor devices andrelated methods of the present invention without departing from thespirit or scope of the invention. Thus, it is intended that the presentinvention cover modifications and variations that come within the scopeof the appended claims and their equivalents.

What is claimed is:
 1. A method for detecting at least one antigen usinga biosensor, the biosensor having a capture chamber and a detectionchamber which are separate from and in fluid communication with eachother, the method comprising: (a) applying a sample in the capturechamber, the sample containing first antigens and first microparticleseach having multiple antibodies immobilized on their surfaces; (b)allowing some of the first microparticles to form aggregates of thefirst microparticles mediated by the first antigens in the capturechamber, wherein the antibodies on the first microparticles bind to thefirst antigens, at least some of the first antigens each bind atmultiple sites to multiple first microparticles simultaneously, andmultiple first microparticles are bound to each other by the firstantigens; (c) separating single first microparticles that did not formaggregates and the aggregates of the first microparticles by retainingonly the aggregates of the first microparticles in the capture chamber;(d) thereafter, injecting an elution buffer into the capture chamber todissociate the aggregates of the first microparticles into single firstmicroparticles and to cause the single first microparticles to move tothe detection chamber; and (e) detecting, in the detection chamber, anamount of single first microparticles obtained from dissociatedaggregates of the first microparticles.
 2. The method of claim 1,wherein the method simultaneously detects the first antigens and secondantigens, wherein in step (a), the sample further contains the secondantigens and second microparticles each having multiple antibodiesimmobilized on their surfaces; wherein step (b) further includesallowing some of the second microparticles to form aggregates of thesecond microparticles mediated by the second antigens in the capturechamber, wherein the antibodies on the second microparticles bind to thesecond antigens, at least some of the second antigens each bind atmultiple sites to multiple second microparticles simultaneously, andmultiple second microparticles are bound to each other by the secondantigens; wherein step (c) further includes separating single secondmicroparticles that did not form aggregates and the aggregates of thesecond microparticles by retaining only the aggregates of the secondmicroparticles in the capture chamber; wherein step (d) further includesdissociating the aggregates of the second microparticles into singlesecond first microparticles, by using an elution buffer that dissociatesboth the aggregates of the first microparticles and the aggregates ofthe second microparticles; and wherein step (e) further includesdetecting, in the detection chamber, an amount of single secondmicroparticles obtained from dissociated aggregates of the secondmicroparticles, wherein the single first microparticles and the singlesecond microparticles generate different detection signals.
 3. Themethod of claim 1, wherein the separating step includes providing afilter disposed at one end of the capture chamber, the filter passingthe single first microparticles and blocking the aggregates of the firstmicroparticles, and washing the aggregates by flowing a wash solutionthrough the capture chamber and the filter.
 4. The method of claim 1,wherein the separating step includes providing two filters disposed atboth ends of the capture chamber, the filters passing the single firstmicroparticles and blocking the aggregates of the first microparticles,and washing the aggregates by alternatingly flowing a wash solutionthrough the capture chamber and the filters in opposite directions. 5.The method of claim 1, wherein the separating step includes providing afilter between the capture chamber and a transfer chamber, the filterpassing the single first microparticles and blocking the aggregates ofthe first microparticles, and circulating the sample containing thesingle first microparticles and the aggregates of the firstmicroparticles in the capture chamber over the filter.